Fig 2: IFNκ increased the efficiency of CAR T cells in vitro. A, Analysis of cytotoxicity of CAR T cells against H460 and H322 cells at the indicated E:T ratios for 16 hours. B, CD69 expression in CAR T cells was analyzed after incubation with H460 cells for 16 hours. C, Release of perforin and GzmB from the supernatant was determined using ELISA after incubation with H460 cells for 16 hours. D, PD1 and TIM-3 expression in CAR T cells was examined after incubation with H460 cells. E, eFlour 670 was analyzed in CAR T cells by incubating the cells with H460 cells for 0 or 7 days, respectively. F, CAR T-cell differentiation was analyzed by incubation with H460 cells. Each value represents the mean ± SD, n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The experiments were independently performed three times, and the results from one representative experiment are displayed. Student t test, and one- and two-way ANOVA were used. E:T, effector to target; GzmB, granzyme B; MFI, mean fluorescence intensity.

Fig 3: Generate HSPC-engineered AlloCAR70-NKT cells with high yield, high purity, and no fratricide risk(A and B) Schematics showing the generation of AlloCAR70-NKT cells (A) and the design of Lenti/iNKT-CAR70-IL-15 lentivector (B).(C) Fluorescence-activated cell sorting (FACS) monitoring of the generation and CD4/CD8 expression of AlloCAR70-NKT cells during the 6-week culture.(D) FACS detection of CAR70 on AlloCAR70-NKT cells. CAR70 was stained using an anti-CD27 monoclonal antibody.(E) Purity of AlloCAR70-NKT cells (n = 6; n indicates different CB donors).(F–J) Study the fratricide risk of AlloCAR70-NKT cells. (F) FACS detection of CD70 on AlloCAR70-NKT cells. Healthy donor PBMC-derived conventional CAR70-T cells were included as a control. (G) Quantification of (F) (n = 6). (H) Fold expansion of the indicated cells normalized to their BCMA-targeting CAR (BCAR)-engineered counterparts (n = 5). (I) Quantification of PD-1+LAG-3+TIM-3+ cells as a percentage of the total cell population. (J) Schematics showing the absence of CD70 expression on AlloCAR70-NKT cells, demonstrating resistance to fratricide and reduced T cell exhaustion.(K) FACS detection of surface marker expression, as well as intracellular cytokine and cytotoxic molecule production of AlloCAR70-NKT and conventional CAR70-T cells.(L–O) Antigen responses of AlloCAR70-NKT cells. AlloCAR70-NKT cells were stimulated with/without αGC-loaded PBMCs for 1 week. (L) Experimental design. (M) Growth curve of AlloCAR70-NKT cells (n = 4). (N) ELISA measurements of effector cytokine levels in the culture supernatants collected on day 5 (n = 4). (O) ELISA measurements of IL-15 levels in the culture supernatants collected at 48 h (n = 4).Representative of over 5 experiments. Data are presented as the mean ± SEM. ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 by Student’s t test (G, H, N, and O) or one-way ANOVA (I).

Fig 4: Design and in vitro characterization of anti-PD-1×IL-10M(A) CD8+ T cells were isolated from PBMCs and induced by CD3/CD28 magnetic beads for 5 days in vitro, as the exhausted CD8+ T cells. The expression of PD-1, TIM-3, or IL-10R was detected by FCM analysis.(B) Schematic diagram of anti-PD-1×IL-10M. IL-10M is linked to the C terminus of the anti-PD-1 antibody light chain via (G4S)4G.(C) Binding of each indicated molecule to CHO-human PD-1 (left) or CHO-human IL-10R cells (right) was analyzed by FCM analysis. MFI means mean fluorescence intensity. Data are representative of three independent experiments.(D) The bioactivity of the indicated proteins was detected in the reporter gene system.(E) IFN-γ secretion in the supernatant was detected by ELISA in SEB (left) or MLR (right) experiments. Statistical analyses were performed by one-way ANOVA with Dunnett’s multiple comparisons tests (ns, not significant; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001). Data are representative of two independent experiments performed on different donors.(F) The Fc region is a hinge-stabilized hIgG1 mutant subtype. Binding of each indicated molecule to C1q was analyzed by ELISA (left), and ADCC activity was detected by reporter gene assay (right). Statistical analyses were performed by two-way ANOVA with Dunnett’s multiple comparisons tests (∗∗∗∗p ≤ 0.0001).Results are reported as the mean ± SEM.

Fig 5: IFNκ increased the efficiency of CAR T cells in vitro. A, Analysis of cytotoxicity of CAR T cells against H460 and H322 cells at the indicated E:T ratios for 16 hours. B, CD69 expression in CAR T cells was analyzed after incubation with H460 cells for 16 hours. C, Release of perforin and GzmB from the supernatant was determined using ELISA after incubation with H460 cells for 16 hours. D, PD1 and TIM-3 expression in CAR T cells was examined after incubation with H460 cells. E, eFlour 670 was analyzed in CAR T cells by incubating the cells with H460 cells for 0 or 7 days, respectively. F, CAR T-cell differentiation was analyzed by incubation with H460 cells. Each value represents the mean ± SD, n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001. The experiments were independently performed three times, and the results from one representative experiment are displayed. Student t test, and one- and two-way ANOVA were used. E:T, effector to target; GzmB, granzyme B; MFI, mean fluorescence intensity.